Project Summary/Abstract Bacterial cells are complex organisms that, like eukaryotic cells, contain different cytoskeletal elements. However, unlike eukaryotic cells, for which much work has been done to determine the role of the cytoskeleton in cellular physiology, there has been very little work on this aspect of bacterial cell biology. Disruption of the cytoskeleton often leads to cell shape changes, making it difficult to separate direct roles of the cytoskeleton from changes due to cell shape defects. This problem has been overcome in my past research by using suppressor mutations that restore cell shape to cell shape mutants without altering other physiological conditions. We will use multiple strategies in this proposal to overcome this problem. The goal of this work is to remove this black box of microbiology, and determine the role of the cytoskeleton in microbial physiology. A deeper understanding of bacterial physiology can guide the development of novel antibacterial therapies, as well as provide insight into the evolution of the cytoskeleton in general. Disrupting basic physiology will make it difficult for infections to take hold in the body, giving the host?s natural defenses time to clear the infection. This proposal will focus on the role of MreB in bacterial motility and chemotaxis. To understand how MreB is needed in chemotaxis we will use a combination of genetic, biophysical, and microscopy techniques to examine the protein dynamics of important components of the chemotaxis system with and without perturbed MreB. We will screen a deletion library for mutants that can bypass the need for MreB in chemotaxis. Additionally, using single-cell techniques, we will measure the run/tumble frequency of cells with and without perturbed MreB. Using known mutations that effect the function of the chemotaxis proteins, we will determine where in the signaling cascade MreB acts. All together this research will determine the role of the bacterial cytoskeleton in bacterial chemotaxis and cell shape. These insights will provide a starting point for the development of novel therapeutics that can be used to stop or slow infections, giving the host immune system time to clear the infection. Additionally, many pathogens use flagella-based motility to increase their virulence. Learning more about how chemotaxis works will enable us to develop therapies that can inhibit motility in the host, lessening infection. This study will be the first to clearly show a role for the bacterial cytoskeleton in cell physiology, independently from cell shape, while adding to our knowledge on both cell shape regulation and chemotaxis.